Signalling and transcriptional regulation of early developmental lineage decisions

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Abstract

Embryonic stem (ES) cells are cell lines isolated from the embryo at a time just prior to implantation
into the uterus. In the right cocktail of medium and cytokines, these cell lines can be maintained
indefinitely in vitro in a self-renewing state. Initially it was assumed that these cells represented a
homogeneous population however, more recently it has been shown that there are a great number of
genes that are expressed heterogeneously. ES cell cultures are therefore a mix of different
subpopulations, some of which have distinct functional properties including a bias or ‘lineage
priming’ towards a particular cell fate. These populations are also dynamic in nature, converting from
one state to another with fairly rapid kinetics.
The main focus of this thesis was to gain a more in depth understanding of the mechanisms regulating
heterogeneity and lineage priming in murine ES cells by asking which signalling pathways play a role
in this phenomenon and how the switch between states is regulated at a transcriptional level. These
questions were asked using an ES cell line containing a sensitive reporter for the endoderm marker
Hex. This reporter, developed by a previous lab member, allowed the identification and separation of
a population of ES cells primed towards a primitive endoderm fate.
Primarily, I assessed the effect of a defined culture system (2i) on the Hex-expressing population. This
culture system contains inhibitors that block FGF signalling and the Wnt pathway component GSK3.
Culturing ES cells in 2i has been suggested to generate a more homogeneous culture. Here, I have
shown that culturing ES cells or pre-implantation embryos in 2i did not eliminate heterogeneity but
maintained them in an early state prior to lineage segregation. When ES cells were cultured in
standard serum-containing medium, Hex was expressed in a mutually exclusive manner with the
embryonic marker NANOG, while in 2i a subpopulation of cells coexpressed both Hex and NANOG.
This population was functionally primed towards extraembryonic endoderm and trophoblast.
Furthermore, these ES cells could efficiently contribute to 2-cell embryos in chimaera assays. LIF
signalling promoted this population through the JAK/STAT pathway.
I then asked how transcription was regulated during the switch between unprimed ES cells to those
primed towards a primitive endoderm fate, as well as how regulation changes during further
differentiation. To ask this, Hex positive (primed) and negative (unprimed) ES cell populations were
sorted as well as a Hex positive differentiated sample. These samples were analysed by GRO-seq to
determine the location, density and orientation of RNA-polymerase throughout the genome. Changes
in gene expression between primed and unprimed states were regulated primarily through elongation
whereas genes upregulated during differentiation were regulated at the point of de novo initiation.